The spread of many diseases depends on the environmental stability of pathogens. While the accumulation of compatible osmolytes inside microbial cells is well understood, there is practically no information about osmolyte efflux systems imparting resistance to abrupt environmental changes. There is also a lack of practical approaches for monitoring membrane perturbations associated with the permeation of lipophilic drugs, which limits our understanding of permeation mechanisms and hampers the process of new drug development. Biophysical and electrophysiological approaches critically help us to decipher membrane mechanisms involved in osmoregulation and to detect interfacial effects of permeant lipophilic substances. Intact bacteria are too small for electrophysiology; however, giant spheroplast preparation provides direct access to the bacterial cytoplasmic membrane. This procedure, involving the induction of filamentous forms by antibiotics followed by cell wall digestion, was developed for E. coli and led to the identification of several mechanosensitive (MS) channels mediating turgor pressure adjustment during osmotic downshock. The mechanosensitive channels of small and large conductance, MscS and MscL, are dominant and gated directly by membrane tension. These channels are also sensitive to lateral pressure changes induced by insertion of lipophilic substances into the surrounding bilayer. Having a substantial amount of information about E. coli channels, in this exploratory project we propose extension of this electrophysiological platform to two common facultative pathogens, Vibrio cholerae and Pseudomonas aeruginosa with the aim of better understanding the mechanoelectrical responses of their membranes, osmolyte transport and lipophilic drug permeation. The genomes of both facultative pathogens contain orthologs of several E. coli MS channels. We will optimize the procedure of giant spheroplast preparation and conduct a detailed study of the mechanosensitive channels in both pathogens. This will include in situ electrophysiological characterization under different pressure stimuli, cloning, homologous and heterologous (E. coli) expression of the channels, determination of their conductances, tension dependences and permeabilities for ions and major compatible osmolytes. It will also include a comparison of the mechano-electrical characteristics in the native membranes and in E. coli. The proposed study will provide us with the first molecular information about the mechanism of an osmotic permeability response in these pathogens, which is a critical environmental defense. Furthermore, understanding how the MS channels function as lateral pressure sensors will open up opportunities for optimizing biologically active compounds such as antibiotics, autoinducers and their synthetic analogs to target specific pathogens by favoring permeation through their cytoplasmic membranes.